Hrp conjugated goat α mouse

Cells were collected in chilled Dulbecco’s Phosphate Buffered Saline (Hyclone) and lysed with sonication in high salt lysis buffer (50 mM Tris, pH 8; 500 mM NaCl; 1% Triton X-100) with protease inhibitors (Complete Mini Protease Inhibitor; Roche). Protein was quantified and separated by SDS-PAGE in a 13% poly-acrylamide gel then transferred to PVDF. Membranes were blocked for at least 1 h in 5% skim milk powder in PBS containing 0.1% Tween-20. Membranes were incubated for at least 2 h in primary antibodies: α-MYC (1:500 monoclonal 9E10 in blocking solution) or anti-β-actin (Genetex at 1:2000) and 1 h in secondary (1:10,000 HRP-conjugated goat-α-mouse, Bio-Rad). Immunoreactive bands were visualized with Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences) and imaged with a Bio-Rad molecular Imager Chemi-Doc XRS+ and ImageLab software (Bio-Rad).

Cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature and permeabilized with 0.05% saponin and 2% BSA in PBS (blocking solution). Primary and secondary antibodies were diluted in blocking solution and DAPI was diluted in PBS before coverslips were mounted using Prolong Gold Antifade (Invitrogen). Images were acquired with a Zeiss LSM700 or LSM710 confocal laser scanning microscope and processed using Fiji [45 (link)]. To measure clathrin intensity, five measurements of intensity were taken at the CCV or in the cytoplasm and averaged. For immunofluorescence microscopy (IF) and western blotting (WB) the following antibodies were used at the designated dilutions: α-clathrin heavy chain (Abcam, WB = 1:2000, IF = 1:1000), α-FCHO2 (ThermoFisher, WB = 1:1000), α-β-actin (Perkin Elmer, WB = 1:5000), polyclonal α-Coxiella (IF = 1:10000), α-LAMP1 (DHSB, IF = 1:250), HRP-conjugated goat α-mouse (BioRad, WB = 1:3000), HRP-conjugated goat α-rabbit (Perkin Elmer, WB = 1:3000), AlexaFluor 488 (ThermoFisher, IF = 1:2000), AlexaFluor 568 (ThermoFisher, IF = 1:2000). For clathrin localization studies, HeLa cells were seeded at 2.5 × 10⁴ per well in 24 well plates with coverslips and the following day infected at an MOI of 100. Cells were incubated for 72 hours and processed as described above.